Lipoxygenase is an enzyme which catalyzes the oxidation of unsaturated fatty acids to hydroperoxides. The oxidation of fatty acids by 5- lipoxygenase is the first step in leukotriene biosynthesis, which has been implicated as a mediator in inflammation and hypersensitivity reactions. Therefore, the inhibition mechanism of lipoxygenase and the development of clinically useful lipoxygenase inhibitors have become the subject of intensive research. One proposed mechanism of the inactivation of lipoxygenase by inhibitors is the reduction of the catalytically active ferric form of the enzyme to its ferrous form. Recent studies have shown that compounds containing the hydroxamate moiety are potent inhibitor is thought to bind to iron at the catalytic site of the enzyme. We now report evidence that the NOH of the hydroxamate group of N-(4-chlorophenyl)-N'-(3- chlorophenyl) area (CPHU), N-[(E)-3-(3-phenoxyphenyl)prop-2-enyl]aceto- hydroxamic acid (BW A4C), and N-(1-benzo(b)thien-2-ylethyl)-N-hydroxyurea (Zileuton) is oxidized by lipoxygenase to form their corresponding nitroxides, which are directly detected by electron paramagnetic resonance spectroscopy. It is consistently found that the selected NOH-containing compounds, e.g., alkylhydroxylamines or N-hydroxyureas, are also oxidized by lipoxygenase to form their corresponding nitroxides. Studies involving a nucleophilic addition by water to a nitrone spin trap, DMPO, were extensively investigated. The HPLC/EPR results demonstrated that the assignment made by previous workers was not correct due to this nucleophilic reaction which partially contributed to the DMPO radical adduct.